Augmented oxidative stress of platelets in chronic smokers. Mechanisms of impaired platelet-derived nitric oxide bioactivity and augmented platelet aggregability.

OBJECTIVES: We investigated whether impaired platelet-derived nitric oxide (PDNO) bioactivity and augmented platelet aggregability in chronic smokers are related to the imbalance of the intraplatelet redox state through increased oxidative stress. BACKGROUND: Chronic smoking impairs PDNO release and augments platelet aggregability. However, their mechanisms are unknown. METHODS: Collagen-induced PDNO release, platelet aggregation, plasma and intraplatelet vitamin C and reduced glutathione (GSH), intraplatelet cyclic guanosine 3',5'-monophosphate (cGMP) and intraplatelet nitrotyrosine production, which is a marker of the peroxynitrite formation, were measured in 11 chronic smokers and 10 age-matched nonsmokers. RESULTS: Release of PDNO and levels of intraplatelet cGMP were lower, and platelet aggregation was greater, in smokers than in nonsmokers. Intraplatelet vitamin C and GSH levels were lower in smokers than in nonsmokers. Intraplatelet nitrotyrosine production was greater in smokers than in nonsmokers. Next, we investigated the effects of oral vitamin C administration (2 g). After vitamin C administration, intraplatelet vitamin C levels were increased and not different at 2 h between the two groups. Then, PDNO release, intraplatelet cGMP levels and platelet aggregation in smokers were restored to the levels of nonsmokers. In smokers, PDNO release and consumption of GSH during platelet aggregation were inversely correlated, and consumption was much less after vitamin C administration. Vitamin C administration decreased intraplatelet nitrotyrosine production in smokers. CONCLUSIONS: Impaired PDNO bioactivity and augmented platelet aggregability may be caused by an imbalance of the intraplatelet redox state through increased oxidative stress in smokers.     

'Spontaneous' platelet aggregation in whole blood in diabetic patients with and without microvascular disease.

Consistent abnormalities of agonist-induced platelet aggregation, in either whole blood or platelet rich plasma, have not been demonstrated in diabetic patients without microvascular disease. In the present study platelet aggregation in the absence of exogenous agonists ('spontaneous' aggregation) was compared between 22 non-diabetic subjects and 23 Type 1 diabetic patients with (n = 12) and without (n = 11) microvascular disease. 'Spontaneous' aggregation was determined by measuring the percentage fall in single platelet number in aliquots of whole blood shaken for 60 min. Diabetic patients without microvascular disease had fewer single platelets remaining (greater aggregation) than non-diabetic subjects at all time-points (69.7 +/- 6.6 vs 82.3 +/- 7.3% at 60 min p less than 0.001), but more platelets remaining than in diabetic patients with microvascular disease at all time-points (69.7 +/- 6.6 vs 61.0 +/- 7.8% at 60 min p less than 0.02). No significant correlations were observed between platelet aggregation and plasma glucose, blood cell counts, or glycated haemoglobin levels. The study suggests that platelet abnormalities antedate the appearance of microvascular disease in diabetic patients.     

Calcitropic hormones, platelet calcium, and blood pressure in essential hypertension

Plasma ionized calcium, platelet cytosolic calcium (using the fura-2 method in gel-filtered platelets), parathyroid hormone (both the intact hormone and a midmolecule portion), calcitriol, and calcidiol were measured in 19 untreated male patients with essential hypertension and 19 age-matched normotensive male research subjects. Mean levels of platelet cytosolic calcium, parathyroid hormone, calcitriol, and calcidiol were all significantly higher, whereas plasma ionized calcium was significantly lower, in the hypertensive group compared with the normotensive group. Both platelet cytosolic calcium and intact parathyroid hormone were positively correlated with mean arterial pressure (r = 0.58, p less than 0.001; r = 0.54, p less than 0.001, respectively), whereas plasma ionized calcium was inversely correlated with mean arterial pressure (r = -0.60, p less than 0.001) in the combined group of all study subjects. All three of these correlations were significant in the hypertensive group alone but not in the normotensive group alone. When analyzed with plasma ionized calcium, body mass index, serum calcitriol, and calcidiol in a multivariable regression model, the significance of the partial regressions of platelet cytosolic calcium and parathyroid hormone with mean arterial pressure persisted. Intact parathyroid hormone was positively correlated to platelet cytosolic calcium (r = 0.43, p less than 0.01) and plasma ionized calcium was inversely correlated to platelet cytosolic calcium (r = -0.44, p less than 0.01). These results confirm previous reports of disturbances of calcium metabolism in essential hypertension and suggest that the elevated platelet cytosolic calcium observed in essential hypertension may be linked to one or more of these alterations of calcium metabolism.     

Intracoronary platelet activation in ischemic heart disease: effects of ticlopidine

Plasma levels of platelet factor 4 have been measured in the aortic and coronary sinus blood of 35 patients: group I (n = 12) with normal coronary arteriograms; group II (n = 15) with angiographically proven coronary artery disease; and group III (n = 8) composed of patients with ischemic heart disease who were being treated with the antiaggregant agent ticlopidine at the time of cardiac catheterization. The mean increase in platelet factor 4 levels through the coronary circulation was 27.4 +/- 21.9 ng/ml (mean +/- standard deviation) in group II, compared with -1 +/- 4.5 ng/ml in group I (p less than 0.01). In group III plasma levels of platelet factor 4 in aortic and coronary sinus samples were all within the normal range. Thus, we conclude that platelet activation constantly occurs in the coronary circulation of patients with stable coronary artery disease, and can be prevented with ticlopidine.     

Regulation of platelet heterogeneity: effects of thrombocytopenia on platelet volume and density

We have examined the effects of variable degrees of acute thrombocytopenia on platelet levels, mean platelet volume (MPV), and buoyant density after induction of thrombocytopenia by platelet antiserum (PAS) in mice with or without spleens. Mice were studied serially 10-16, 36, 48, 60-64, 84, 108, 144, 180, 228, 276, 348-360, 372, and 516 h after PAS treatment. MPV and platelet count (PC) x 10(6)/microliters for normal intact mice (n = 136) were 4.7 +/- 0.3 fl (SD) and 1.69 +/- 0.52 (SD), respectively. Twelve hours after PAS-induced severe thrombocytopenia (PC less than 0.05 x 10(6)/microliters), MPV increased significantly (p less than 0.01) to 6.4 fl, was maximal at 36 h (8.2 fl), remained elevated until 144 h following PAS treatment, and then returned to normal. Platelet density decreased significantly (p less than 0.05) 64 h after PAS treatment and returned to normal at 144 h. Hematocrits of repeatedly bled intact control mice decreased from 45% to 30%, accompanied by thrombocytosis (maximal PC 2.24 x 10(6)/microliters) without significant changes in either MPV or platelet density. Moderate thrombocytopenia (PC 0.1-0.2 x 10(6)/microliters) in intact mice produced significantly (p less than 0.05) increased MPV, at 5.7 fl 12 h after PAS treatment, with a peak MPV of 7.6 fl (p less than 0.001) at 36 h; MPV returned to normal at 84 h. Platelet density decreased (p less than 0.001) 12 h after PAS treatment and returned to baseline at 228 h. Control splenectomized mice (n = 185) had an MPV of 5.0 fl +/- 0.7 fl and a PC of 2.14 +/- 0.6 x 10(6)/microliters. Comparably severe and moderate thrombocytopenia in splenectomized mice produced alterations in platelet count, MPV, and density similar to those in intact mice, although maximal MPV and the degree of rebound thrombocytosis after severe thrombocytopenia were more marked in splenectomized mice. In response to reduction of the platelet mass in both intact and splenectomized mice, MPV increased in proportion to the severity of thrombocytopenia, occurred as early as 4 h after induction, and persisted during early rebound thrombocytosis. Previous observations that megakaryocyte ploidy did not shift until 48 h after onset of thrombocytopenia confirm that both initial and maximal changes in MPV in response to this stimulus are regulated by processes other than alterations of megakaryocyte DNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vitamin C in the human stomach: relation to gastric pH, gastroduodenal disease, and possible sources.

Fasting gastric juice pH and concentrations of vitamin C in gastric aspirate and plasma were measured in 73 patients undergoing endoscopy. Vitamin C concentrations were significantly lower in those with hypochlorhydria (pH greater than 4; n = 23) compared with those with pH less than or equal to 4 (p less than 0.005) and there was a significant correlation between gastric juice and plasma concentrations (p = 0.002). Patients with normal endoscopic findings had significantly higher intragastric concentrations of vitamin C than those with gastric cancer (p less than 0.001), pernicious anaemia (p less than 0.005), gastric ulcer (p less than 0.01), duodenal ulcer (p less than 0.05), or after gastric surgery (p less than 0.01). There was a strong trend (0.05 less than p less than 0.1) towards lower intragastric concentrations of vitamin C in patients with chronic atrophic gastritis. In vitro, vitamin C concentrations remained stable in acidic but fell significantly over 24 hours in alkaline gastric aspirate. Gastric secretory studies in five volunteers showed that vitamin C concentrations increased significantly after intramuscular pentagastrin. These findings suggest that the low fasting levels of vitamin C in hypochlorhydric gastric juice may be caused by chemical instability and that vitamin C may be secreted by the human stomach.     

Plasma concentrations of platelet-specific proteins correlated with platelet survival

Relationships between 51Cr platelet survival and plasma concentrations of beta-thromboglobulin (betaTG) and platelet factor 4 (PF4) were analyzed in 91 studies of patients with coronary artery disease. betaTG was significantly correlated with platelet life-span, turnover, and the number of hits in the multiple hit model. PF4 was significantly correlated with life-span and turnover. The most significant relationship involving platelet-specific protein concentrations and life-span estimates was between betaTG and life-span estimated using the multiple hit model (r = -0.39, p less than 0.001). There was a high correlation between betaTG and PF4 (r = 0.62, p less than 0.001), and no improvement could be obtained by combining the measurements of the two proteins in any regression with life-span or turnover. The results indicate that the patients with the shortest platelet survival time in this group tended to have the highest plasma concentration of betaTG and PF4 and thus probably increased in vivo release of betaTG and PF4. They strengthen the claim that these platelet-specific proteins may be indicators of platelet involvement in disease.     

Fibrinopeptide A, platelet factor 4, and beta-thromboglobulin levels in coronary heart disease

In vivo platelet alpha-granule release and fibrin I formation were measured in 82 patients with ischemic heart disease by radioimmunoassay of platelet factor 4, beta-thromboglobulin, and fibrinopeptide A. The presence and extent of coronary artery disease were determined by coronary arteriography, and the extent of left ventricular regional dysfunction was assessed by contrast left ventriculography. In patients with abnormal coronary arteriograms without previous myocardial infarction, mean levels of platelet factor 4, beta-thromboglobulin, and fibrinopeptide A were not elevated. In patients in whom myocardial infarction had occurred more than 6 mo previously, platelet factor 4 (8.3 ng/ml; p less than 0.01) and beta-thromboglobulin (33.2 ng/ml; p less than 0.001) levels were significantly elevated, but fibrinopeptide A levels were normal. Levels of platelet factor 4 and beta- thromboglobulin were unrelated to the extent of coronary artery disease. In the patients with prior infarction, beta-thromboglobulin correlated directly with extent of left ventricular regional dysfunction (r = 0.53; p less than 0.01) and inversely with ejection fraction (r = -056; p less than 0.05). In a small group of patients with left ventricular aneurysm, mean fibrinopeptide A levels were also elevated. We interpret these findings as indicating that platelet release in patients with ischemic heart disease results from platelet reaction with previously infarcted myocardium rather than with the atherosclerotic coronary arteries.     

Importance of increased urinary calcium excretion in the development of secondary hyperparathyroidism of patients under glucocorticoid therapy

Parathyroid function and calcium metabolism were studied in 44 patients under glucocorticoid therapy (steroid group) and in 25 control subjects. Nephrogenous cAMP and serum immunoreactive parathyroid hormone levels in the steroid group were significantly higher than those in control subjects (p less than 0.001). Nephrogenous cAMP in the steroid group correlated positively with prednisolone dosage (r = 0.424, p less than 0.01), and most patients who showed obvious elevations of nephrogenous cAMP had received over 10 mg/day of prednisolone for at least 2 mo. Fasting urinary calcium in the steroid group [166.1 +/- 78.5 (+/- SD) mg/g creatinine] was about 2 times greater than that in control subjects (74.1 +/- 35.6) (p less than 0.001). Fasting urinary calcium in control subjects correlated negatively with nephrogenous cAMP (r = -0.486, p less than 0.02). In contrast, these values in steroid group showed significant positive correlation (r = 0.631, p less than 0.001), suggesting that increased urinary calcium excretion is an important factor in the development of secondary hyperparathyroidism. Elevated nephrogenous cAMP and serum immunoreactive parathyroid hormone levels decreased after the administration of trichlormethiazide and/or 1 alpha hydroxy-vitamin D3. We conclude that increased urinary calcium excretion plays an important role in the development of secondary hyperparathyroidism in patients under glucocorticoid therapy and that the administration of thiazide and/or vitamin D could improve the secondary hyperparathyroidism caused by glucocorticoid therapy.     

A monoclonal antibody against the platelet glycoprotein IIb/IIIa receptor complex prevents platelet aggregation and thrombosis in a canine model of coronary angioplasty.

BACKGROUND. The comparative effects of aspirin and F(ab')2 fragments of monoclonal antibody 7E3 against the platelet glycoprotein IIb/IIIa receptor on ex vivo platelet aggregation and in vivo thrombosis were studied in a canine coronary balloon angioplasty model. METHODS AND RESULTS. Three groups were studied. Group 1 (n = 8) was pretreated with saline placebo, group 2 (n = 8) was pretreated with 325 mg aspirin, and group 3 (n = 8) was pretreated with 0.8 mg/kg 7E3 F(ab')2. Coronary angioplasty was performed in the left anterior descending coronary artery of open-chest dogs under fluoroscopic control; serial measurements of basal and hyperemic coronary blood flows were then made for 2 hours after application of an external stenosis that decreased hyperemic flow by 50%. There were no significant differences in platelet counts or hemodynamic measurements during the experiments. Platelet aggregation was decreased by treatment: group 1, 64 +/- 13% versus 50 +/- 13% (p = NS); group 2, 57 +/- 4% versus 25 +/- 4% (p less than 0.001); and group 3, 77 +/- 5% versus 10 +/- 6% (p less than 0.0002). Compared with initial measurements, the 7E3 antibody was superior to aspirin in maintaining hyperemic coronary blood flow after release of the external stenosis: group 1, 177 +/- 14 versus 21 +/- 14 ml/min (p less than 0.0003); group 2, 189 +/- 9 versus 110 +/- 28 ml/min (p less than 0.008); and group 3, 194 +/- 12 versus 181 +/- 15 ml/min (p less than 0.02). In group 1, arterial occlusion developed in five dogs, and nonocclusive thrombus was seen in three dogs. In group 2, arterial occlusion developed in one dog, and nonocclusive thrombus was seen in five dogs. No thrombotic material was visualized in group 3 dogs treated with 7E3 F(ab')2. CONCLUSIONS. In this animal model, the 7E3 antiplatelet antibody is superior to aspirin in inhibiting platelet aggregation, thrombosis, and acute closure after deep arterial injury caused by coronary balloon angioplasty.     

Heparin-releasable platelet factor 4 in patients with coronary artery disease.

Recently, platelet factor 4 (PF4) release by heparin (heparin-releasable PF4) has been examined as a useful marker of the interaction between the substances liberated from circulating platelets and the vascular endothelium. We compared the plasma levels of PF4 and beta-thromboglobulin (beta-TG) after intravenous heparin injection in patients with coronary artery disease (CAD) and normal control subjects. We also studied the effects of low-dose aspirin (81 mg/day) on the plasma level of heparin-releasable PF4 in the CAD patients. Blood samples were obtained before and 5 min after the intravenous injection of heparin (1,000 IU) from 23 patients with CAD and 15 normal control subjects. Although the plasma beta-TG level remained unchanged after heparin injection, the plasma PF4 level markedly increased in both groups. There was a significant difference in plasma PF4 levels at 5 min after heparin injection between the CAD group (100.1 +/- 38.1) and the control group (61.0 +/- 24.0) (p less than 0.01). The PF4/beta-TG ratio after heparin injection was also higher in the CAD group than in the control group (p less than 0.01). There was a correlation between the PF4/beta-TG ratio after heparin and the Gensini CAD score, which defines the severity of coronary atherosclerosis (r = 0.489, n = 23, p less than 0.01). Low-dose aspirin was administered to 11 CAD patients for 246.0 +/- 28.8 days. Blood samples for the assay of PF4 and beta-TG were obtained as stated above, and platelet aggregation, thromboxane B2 (TxB2), and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) levels were also measured before and during aspirin administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet aggregates as markers of platelet activation: Characterization of flow cytometric method suitable for clinical applications

The present paper describes a flow cytometric method for assay of platelet aggregates (PAg) in blood. This method combines and simplifies previously reported techniques, simultaneously enumerating PAg formed upon platelet activation, their expression of activation marker CD62P (P-selectin), and their content of bound leukocytes (LPAg). The sensitivity of this method to low levels of agonists (ADP, collagen) is compared to conventional aggregometry and some features of platelet-leukocyte interaction are explored. The results were: (1) ADP or collagen induced a dose-dependent increase in PAg number and corresponding decline in free platelets. The ED₅₀ for ADP (0.15 μM) and for collagen (0.2 μg/mL) was about 1/20 the ED₅₀ found by aggregometry, indicating 20-fold greater sensitivity. (2) At higher concentrations, the fraction of PAg with bound leukocytes (LPAg) increased to 60–70%. This rise correlated with PAg size and CD62P expression, but not with the number of PAg formed. (3) The response of whole blood (WBD) to agonists was qualitatively different from that of platelet-rich plasma (PRP): in WBD the population of CD62P+ PAg was much higher than in PRP and the population of CD62P+ free platelets was much lower. This implies that leukocytes rapidly recruit activated platelets. (4) Desmopressin (DDAVP) at 5 nmol/L induced a significant rise in activated (CD62P+) PAg and platelets, even though no effect of DDAVP could be detected by conventional aggregometry; this further confirms that DDAVP acts directly on platelets. (5) Plasma samples from TTP patients induced a rise in PAg when added to normal PRP, though little or no effect could be detected by aggregometry. In summary, the flow cytometric method described here appears useful for detecting low levels of platelet activation and provides information on platelet leukocyte interaction, potentially important in identifying and differentiating thrombogenic states. Since it is rapid and economical, it is well suited for clinical implementation. Am. J. Hematol. 57:33–42, 1998. © 1998 Wiley-Liss, Inc.     

Parathyroid hormone, platelet calcium, and blood pressure in normotensive subjects

Relations between platelet cytosolic calcium, parathyroid hormone, and blood pressure were investigated in 91 normotensive subjects: 47 men and 44 women ranging in age from 24 to 70 years. The men had higher mean arterial blood pressure, serum creatinine, and body mass index than the women. Serum total calcium, plasma ionized calcium, and parathyroid hormone (measured as both intact hormone and mid-molecule fragment) were not different between men and women; however, serum phosphate was higher in women than in men. Basal platelet cytosolic calcium was higher in men than in women (113.7 +/- 1.9 versus 105.9 +/- 1.7, respectively; p less than 0.01), but there was no difference in the peak platelet cytosolic calcium responses to thrombin between the two groups. In the combined group of male and female subjects, platelet cytosolic calcium correlated with diastolic blood pressure and mean arterial pressure (r = 0.37, p less than 0.001 and r = 0.32, p less than 0.01, respectively). Intact parathyroid hormone correlated with systolic and mean arterial blood pressure (r = 0.41, p less than 0.001 for both). Age correlated with both systolic blood pressure (r = 0.40, p less than 0.001) and intact parathyroid hormone (r = 0.51, p less than 0.001). When multiple regression analysis was performed using mean arterial pressure as the dependent variable, platelet cytosolic calcium and intact parathyroid hormone maintained significant correlations with mean arterial pressure. Platelet cytosolic calcium did not correlate with intact parathyroid hormone. These results suggest that both platelet cytosolic calcium and intact parathyroid hormone are associated with blood pressure regulation in normotensive subjects. However, the influences of these two factors on blood pressure are not interrelated.     

Effects of platelet glycoprotein IIb-IIIa number and glycoprotein IIIa Leu33Pro polymorphism on platelet aggregation and sensitivity to glycoprotein IIb-IIIa antagonists

We investigated the influence of glycoprotein (GP) IIIa Leu33Pro polymorphism, platelet GP IIb-IIIa number, and plasma fibrinogen concentration on platelet aggregation and antiaggregatory action of GP IIb-IIIa antagonists. Healthy volunteers with GP IIIa Pro33(-) (Leu33Leu33, n = 20) and Pro33(+) (Leu33Pro33, n = 13, and Pro33Pro33, n = 2) genotypes were included into the study. GP IIIa Leu33Pro substitution was associated with the increase of the level and rate of platelet microaggregate formation induced by GP IIb-IIIa activating antibody CRC54 (100, 200, 400 microg/ml) against the epitope within 1-100 residues of GP IIIa N-terminal part (p from 0.001 to 0.047). No significant differences were detected between parameters of platelet aggregation induced by ADP (1.25, 2.5, 5.0, 20 microM) in GP IIIa Pro33(+) and Pro33(-) donors. GP IIb-IIIa antagonist Monafram (F(ab')(2) fragment of GP-IIb-IIIa blocking antibody CRC64) (1, 2, 3 microg/ml), but not eptifibatide (50, 100, 150 ng/ml) inhibited ADP-induced aggregation slightly less efficiently in GP IIIa Pro33(+) group (p < 0.05 at 1 and 2 microg/ml Monafram). GP IIb-IIIa number (evaluated as maximal binding of (125)I-labelled antibody CRC64) varied from 40.5 to 80.8 x 10(3) per platelet with no significant influence of GP IIIa genotype. Consistent correlations were revealed between GP IIb-IIIa quantity and the level and rate of ADP-induced aggregation (r from 0.353 to 0.583, p from <0.001 to 0.037) as well as resistance (level of residual aggregation) to both GP IIb-IIIa antagonists (r from 0.345 to 0.602, p from <0.001 to 0.042). ADP-induced aggregation was considerably increased and efficiency of GP IIb-IIIa antagonists decreased in donors with high in comparison with low GP IIb-IIIa quantity (>60 and 40-50 x 10(3) per platelet respectively, p < 0.01 for most tests). No correlations were observed between all tested parameters and plasma fibrinogen concentration. Our results indicate that inter-individual variability of platelet GP IIb-IIIa number significantly affects platelet aggregation and antiaggregatory effects of GP IIb-IIIa antagonists. Contribution of this factor is higher than that of GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration.     

Treatment of severe platelet dysfunction and hemorrhage after cardiopulmonary bypass: reduction in blood product usage with desmopressin

Impairment of platelet function commonly occurs after cardiopulmonary bypass, and may result in substantial bleeding. Because desmopressin acetate (a synthetic analogue of vasopressin) shortens bleeding time in a variety of platelet disorders, a controlled clinical trial of intravenous desmopressin was performed in 39 patients with excessive mediastinal bleeding (greater than 100 ml/h) and a prolonged template bleeding time (greater than 10 minutes) more than 2 hours after termination of cardiopulmonary bypass. Twenty-three desmopressin recipients and 16 control patients (no desmopressin) were similar in surgical procedure, pump time, platelet count, template bleeding time and amount of bleeding before therapy (p = NS). Compared with the control group, the patients receiving desmopressin (20 micrograms; mean 0.3 micrograms/kg) utilized fewer blood products (29 +/- 19 versus 15 +/- 13 units/patient; p less than 0.05), especially platelets (12 +/- 9 versus 4 +/- 7 units/patient; p = 0.004), while achieving a similarly effective reduction in mediastinal bleeding (4.8- and 4.3-fold, p = 0.001 for both). Severe platelet dysfunction was partially corrected within 1 hour after desmopressin infusion, during which interval no blood products were administered: the template bleeding time shortened (from 17 to 12.5 minutes, p less than 0.05), whereas the platelet count remained unchanged (at 96 +/- 35 and 105 +/- 31 X 10(3)/mm3, p = NS). The plasma levels of two factor VIII components increased: procoagulant activity (VIII:C) from 0.97 +/- 0.43 to 1.52 +/- 0.74 units/ml (p less than 0.05) and von Willebrand factor (VIII:vWF) from 1.28 to 1.78 units/ml (p less than 0.05); these increases correlated with the shortening of the bleeding time (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-associated immunoglobulins IgG, IgM, IgA and complement C3c in chronic idiopathic autoimmune thrombocytopenia: relation to the sequestration pattern of 111indium labelled platelets

Levels of platelet-associated immunoglobulins (PAIg) IgG, IgM, IgA and complement C3c were related to parameters of 111Indium-labelled platelet kinetics in 17 patients with chronic idiopathic autoimmune thrombocytopenia (cAITP). Elevated levels of PAIg/C3c were found in 14 patients (82%) (PAIgG n = 13, PAIgM n = 11, PAIgA n = 1, PAC3c n = 5). Only PAIgG correlated with platelet counts (RS = -0.71, p less than 0.01). Mean platelet life span (MLS) was shortened in all patients (median 12.0 h, range 0.3-45.6 h) and correlated with the platelet counts (RS = 0.49, p less than 0.05). MLS was correlated with PAIgG (RS = -0.52, p less than 0.05), but not with PAIgM, PAIgA, or PAC3c. The site of sequestration was splenic in 10 patients and splenic-hepatic in 7 patients. Although no significant correlation between either site of platelet sequestration and any of the investigated PAIg/C3c was demonstrable, platelets coated with higher PAIgG levels were more readily sequestrated in the spleen, while elevations of PAC3c were found in 4 out of 7 patients with hepatic involvement.     

The effect of liver disease on factors V, VIII and protein C

The components of the factor VIII complex were estimated by immuno- and bioassays in 85 patients with liver disease. The plasma concentrations of the antigens were elevated in 65% (VIII:CAg) and in 76% (VIIIR:Ag) of patients while the biological activities were elevated in only 14% (VIII:C) and 15% (VIII:RiCof). There was no correlation with C-reactive protein, used as a measure of an acute phase reaction (X2 = 0.7; P = 0.1); or with severity of liver disease as judged by prothrombin ratio (P = 1.0) but highest values were observed in patients with cholestatic liver disease. Following parenteral vitamin K there was a significant fall in both the biological activity of VIIIC (36%) and of VIII:CAg (38%) in 13 vitamin K deficient patients (P less than 0.001) but no change in 23 vitamin K replete patients or in the VIIIR:Ag levels in either group. Factor V levels were lower in patients with parenchymal liver disease (0.54 +/- 0.1 units/ml, mean +/- SEM, n = 12; normal range 0.5-1.5 units/ml) than in patients with extrahepatic cholestasis who were vitamin K deficient (1.2 +/- 0.1 units/ml, P less than 0.0001). The levels of protein C antigen, the vitamin K dependent protease which inactivates factors VIII:C and V, was at the lower end of the range in both groups (0.7 +/- 0.1, mean +/- SEM, n = 18, normal range 0.74-1.4 units/ml). There was no significant change in either protein C antigen or factor V following vitamin K. The discrepancy between the biological activity of factor VIII and the antigen levels could represent accumulation of partially degraded factor VIII or production of a hypoactive form. There is no evidence that the reduction in VIIIC and VIII:CAg following vitamin K was mediated by protein C.     

Elevated levels of circulating platelet aggregates and beta-thromboglobulin in scleroderma

Levels of circulating platelet aggregates and plasma beta-thromboglobulin reflect the degree of platelet activation in vascular injury and repair. Both values were evaluated in 38 patients with scleroderma and 18 control subjects matched for age, sex, and race. Circulating platelet aggregates (expressed as percentage of total platelet count) were 7.2 +/- 5.5% (mean +/- SD) in the control group and 31.2 +/- 13% in the scleroderma group (p less than 0.0005). Beta-thromboglobulin levels in the control groups were 20 +/- 10 ng/mL and 72.7 +/- 50 ng/mL in the group with scleroderma (p less than 0.0005). A positive correlation was found between the two values (r = 0.6, p less than 0.0005). Significant reductions in circulating platelet aggregates and beta-thromboglobulin levels were achieved in 10 patients by dipyridamole and aspirin therapy. These results show in-vivo activation of platelets in scleroderma with release of platelet granule constituents. Antiplatelet therapy in adequate doses returned both values to normal; however, its long-term effect on scleroderma is not yet known.     

Inverse relationships between coronary blood flow obstruction and platelet reactivity in stable angina pectoris

This study investigates relationships between platelet reactivity and coronary blood flow obstruction in stable angina pectoris. Consented were 36 patients with single-vessel disease. The subjects were divided into two groups. One group (n=14) had less severe (<=80%) and the second group (n=22) had severe coronary flow impairment (90%). Before elective coronary angiography platelet in vitro reactivity in venous whole blood was determined using a flow cytometry technique. A thrombin-receptor activating peptide (TRAP-6) (0.77 and 0.06 g/l) and ADP (8.5 and 1.7 micromol/l) were used to activate platelets. The number of fibrinogen positive cells (%) i.e., activated platelets after stimulation was employed as experimental parameter. Less severe flow obstruction was associated with more reactive platelets. When stimulating with 0.77 g/l TRAP-6 the number of activated platelets was 64+/-15 (SD)%. The corresponding value for the group with severe flow obstruction was 40+/-17(SD)%. The difference is significant (P<0.001). 0.06 g/l TRAP-6 yielded similar results (P<0.01). Also when using 8.5 micromol/l ADP to challenge platelets less severe flow obstruction was associated with enhanced reactivity (P<0.01). 1.7 micromol/l ADP generated comparable results (P<0.05). Thus, in stable angina pectoris coronary flow obstruction is inversely related to platelet reactivity.     

Bleeding due to thrombocytopenia in acute leukemias and reevaluation of the prophylactic platelet transfusion policy

Prophylactic platelet administration is indicated at counts below 20 X 10(9)/l. The bleeding tendency and severity were compared between thrombocytopenic patients with acute-lymphocytic leukemia (ALL) and acute non-lymphocytic leukemia (ANLL) in the ranges of 10-20 X 10(9)/l platelets, while prophylactic platelet administration was given only below 10 X 10(9)/l. The bleeding tendency for ALL was quite similar at platelet counts above or below 10 X 10(9)/l. The bleeding tendency was significantly lower (p less than 0.001) when the platelets were above this level in ANLL patients. When the thrombocytopenia was caused by chemotherapy, the bleeding was significantly lower in both types of leukemia above 10 X 10(9)/l (p less than 0.05 for ALL, p less than 0.001 for ANLL) as compared with lower counts. When the thrombocytopenia was caused by leukemia, the bleeding tendency was similar in both types of leukemia and at all platelet counts (below 20 X 10(9)/l). Fever, not associated with sepsis, augmented the bleeding severity of patients with ANLL. Stable or rising counts of platelets were associated with significantly lower bleeding tendency above 10 X 10(9)/l only in ANLL patients. The decision for prophylactic platelet administration at counts below 20 X 10(9)/l should be guided by the type of the leukemia (ALL vs. ANLL), the cause of thrombocytopenia (chemotherapy vs. leukemia per se), the trend of the platelet counts, presence of fever and patient's age (below or above 18 years).(ABSTRACT TRUNCATED AT 250 WORDS)